Project Summary/Abstract Adenosine Deaminases that act on RNA (ADARs) are a family of RNA editing enzymes that convert adenosines to inosines in double-stranded RNA (dsRNA). ADARs are essential in mammals and required for the health of all animals. An important function of ADARs is to target codons to allow a single mRNA to give rise to multiple protein isoforms. Surprisingly, in all studied organisms, recoding editing sites are rare compared to the extensive, non-selective, editing that occurs in noncoding regions of mRNAs, such as introns and untranslated regions. The functions of non-selective editing are unknown, and the proposed research will capitalize on technological advances, such as RNAseq and CRISPR protocols, to fill this gap in knowledge. Studies will utilize the model organism C. elegans, since non-selective editing may be solely responsible for phenotypes of C. elegans lacking ADARs. Experiments will test a model whereby non-selective editing precludes Dicer processing of dsRNA, thus regulating gene silencing in a developmental and tissue-specific manner. Studies are facilitated by the genome-wide identification of Editing-Enriched Regions (EERs), which define the non-selectively edited dsRNAome. RNAseq analyses will define expression of EER-associated genes, and endogenous siRNAs (endo-siRNAs) matching these genes, to reveal genes that are good candidates for ADAR regulation of gene silencing. Deletion of the EER with CRISPR protocols will allow confirmation that ADAR regulation depends on the presence of the EER. Endo-siRNAs aligning to EERs have features of direct Dicer products, a species of small RNA not previously observed among C. elegans endo- siRNAs. Factors involved in the biogenesis of, and silencing by, this newly identified class of endo-siRNAs will be identified with a screen using a C. elegans strain expressing a GFP reporter that is silenced in an ADAR mutant strain. Proposed studies will yield strains that lack double-stranded EER(s) in specific genes, and ADAR mutant strains with additional deletions in factors that rescue ADAR-regulated silencing. These strains will be tested for their ability to rescue phenotypes of C. elegans lacking ADARs. While correlation of a phenotype to a specific gene will be a priority, a degron protocol will provide an alternate strategy. Studies are designed to provide long-awaited details about the functions of C. elegans ADARs, and pave the way for defining similarities and differences in roles of non-selective editing in other organisms.